Diabetes is an impaired metabolic response to our body's own insulin so that active muscle cells cannot take up glucose as easily as they should. When diabetes, or reduced insulin sensitivity, exists, the body attempts to overcome this resistance by secreting more insulin from the pancreas. In that physiologic circumstance, the blood insulin levels are chronically higher which inhibits fat cells from releasing their energy stores to allow for weight loss. Diabetes is associated with obesity, hypertension, abnormal triglycerides and glucose intolerance. BCL-2 family proteins, such as BAD, are well known to play critical roles in organism homeostasis by regulating programmed cell death or apoptosis. In recent years, novel non-apoptotic functions have been identified for select BCL-2 family members and these newly identified roles are vital to maintaining organism homeostasis. Deregulation of the apoptotic functions of BCL-2 family members can lead to excessive cell loss or excessive cell survival, giving rise to diseases such as neurodegeneration and cancer, respectively. Deregulation of the non-apoptotic functions of BCL-2 family members, such as BAD, can produce a diabetogenic phenotype that is unrelated to BAD's role in cell death physiology. The ability of select BCL-2 family proteins to toggle between distinct functions is a critical aspect of their physiologic activity. The molecular mechanisms by which they achieve dual roles can be mediated by their bioactive BH3 domain and, specifically, its phosphorylation state. Therefore, the ability to generate selective compounds that mimic phosphorylated BH3 domains has significant therapeutic potential in treating human disease.
BCL-2 Family Proteins: Critical Intra-Cellular Checkpoints of Apoptosis
Programmed cell death is a genetically conserved pathway essential for proper embryonic development and the maintenance of tissue homeostasis (Cory, S. and Adams, J. M. 2002. Nat Rev Cancer 2, 647-56). Aberrant regulation of this pathway participates in the genesis of multiple human diseases, including cancer, autoimmunity, neurodegenerative disorders, and diabetes. The mammalian apoptotic pathway provides evidence for the participation of organelles, especially mitochondria (Green, D. R. and Kroemer, G. 2004. Science 305, 626-9). Besides providing most of the cellular ATP, mitochondria participate in apoptosis by releasing cytochrome c and other apoptogenic factors. Once released, cytochrome c is assembled together with APAF-1 and caspase-9 to form the “apoptosome”, which in turn activates downstream caspases, leading ultimately to cellular demise (Li, P. et al., 1997. Cell 91:479-89). Mitochondria are also responsible for cellular respiration and coordinate multiple metabolic pathways, yet the interrelationship of these functions with apoptosis has remained uncertain.
The BCL-2 family of proteins constitutes a critical control point in apoptosis residing immediately upstream to irreversible cellular damage, where the members control the release of apoptogenic factors from mitochondria (Danial, N. N. and Korsmeyer, S. J. 2004. Cell 116:205-19). Several Bcl-2 proteins reside at sub-cellular membranes, including the mitochondrial outer membrane, ER and nuclear membranes. The family consists of both death agonists and antagonists, which share sequence homology within one or more segments known as BCL-2 homology (BH) domains (FIG. 1). All anti-apoptotic members, such as BCL-2 and BCL-XL, and a subset of pro-apoptotic family members, such as BAX and BAK, are “multi-domain” proteins sharing sequence homology within 3-4 BH domains. The “BH3-only” subset of pro-apoptotic molecules, including BAD, BID, BIM, NOXA and PUMA show sequence homology only within a single α helical segment, the BH3 domain, which is also known as the critical death domain (Wang, K. et al., 1996. Genes Dev 10:2859-69). BAX and BAK constitute a requisite gateway to the mitochondrial pathway of apoptosis in that cells doubly deficient for these proteins are resistant to all apoptotic stimuli that signal through the intrinsic pathway (Lindsten, T. et al., 2000. Mol Cell 6, 1389-99; Wei, M. C. et al., 2001. Science 292, 727-30). All BH3-only molecules operate upstream of BAX and BAK connecting proximal death and survival signals to the core apoptotic pathway (FIG. 2) (Cheng, E. H. et al., 2001. Mol Cell 8, 705-11; Zong, W. X. et al., 2001. Genes Dev 15, 1481-86). Upon receipt of death signals, BAX and BAK undergo allosteric activation at the mitochondria, resulting in permeabilization of the outer membrane and release of cytochrome c Wei, M. C. et al., 2000. Genes Dev 14, 2060-71) (FIG. 2).
The balance between anti- and pro-apoptotic sub-classes of BCL-2 molecules sets a “rheostat” that determines death susceptibility (Oltvai, Z. et al., 1993. Cell 74, 609-19). BH3-only pro-apoptotic molecules like BAD, BID, BIM actively adjust this “rheostat” and their function is dynamically regulated by distinct mechanisms, including transcriptional control and post-translational modifications. For example, cytosolic BID is activated upon cleavage by caspase-8, leading to mitochondrial translocation, BAX/BAK activation, and cytochrome c release (Li, H. et al., 1998. Cell 94, 491-501; Luo, X. et al., 1998. Cell 94, 481-90 1998). NOXA and PUMA are transcriptional targets of p53 with select roles in apoptosis induced by genotoxic stress (Nakano, K. et al., 2001. Mol Cell 7, 683-94; #328; Yu, J. et al., 2001. Mol Cell 7, 673-82). Additionally, BAD's pro-apoptotic activity is inhibited by phosphorylation in response to extra-cellular growth or survival factors (Zha, J. et al., 1996. Cell 87, 619-281996).
While in vitro studies show that overexpression of BH3-only molecules leads to apoptosis in a variety of cell lines, loss of function mouse models indicate that individual BH3-only proteins serve as cell death initiators responding to selected signals in restricted cell types. The cell type and signal-specific in vivo function of BH3-only molecules suggest that either the functional redundancy of these molecules is tissue type specific, or that BH3-only molecules may have distinct roles in other pathways. Indeed, recent discoveries have unraveled physiologic roles for BCL-2 family proteins beyond apoptosis. The following are but four examples of the integration of apoptosis with cellular homeostatic pathways.
(i) The BCL-2 Family Proteins and Cellular Metabolism:
We previously conducted a proteomic analysis of liver mitochondria, which revealed that the BCL-2 family protein BAD resides in a glucokinase (GK)-containing complex that regulates glucose driven whole cell respiration (Danial N. N. et al., 2003. Nature 424, 952-6) (FIG. 4). The Bad-null genetic model showed that BAD is needed for complex assembly and the non-phosphorylatable Bad 3SA knockin mouse model provided evidence that phosphorylated BAD is required for full mitochondria-tethered GK activity. Consistent with a role for BAD in supporting GK activity, both the Bad-deficient and the Bad 3SA animals display abnormal glucose homeostasis marked by fasting hyperglycemia and glucose intolerance. Because Bad-null and Bad 3SA represent loss and gain of function models for the pro-apoptotic activity of BAD, respectively, the common metabolic abnormalities in these animals suggested that the role of BAD in glucose metabolism may be distinct from its capacity to sensitize cells to apoptosis. Indeed the experiments presented in this application show specific roles for BAD in both pancreas and liver, each with significant physiologic consequences.
(ii) The BCL-2 Family Protein BID and Cellular Response to DNA Damage:
In response to DNA damage, cells either arrest from proliferation to allow sufficient time to repair their DNA or undergo apoptosis. The pro-apoptotic protein BID functions in both apoptosis and in DNA damage checkpoints within cells (Zinkel, S. et al., 2006. Cell Death Differ 13, 1351-9). Loss of BID results in genomic instability and myeloid malignancies (Zinkel, S. et al., 2003. Genes Dev 17, 229-39). BID is a substrate for DNA damage kinase ATM in the nucleus and modulates the intra-S phase checkpoint within cells with damaged DNA (Zinkel, S. et al., 2005. Cell 122, 579-91; Kamer, I. 2005. Cell 122, 593-603). Importantly, while the BH3 domain of BID is required for its apoptotic function at mitochondria, it is dispensable for its cell cycle checkpoint function in the nucleus (Zinkel, S. et al., 2005. Cell 122, 579-91) (FIG. 68).
(iii) The Cross Talk Between Protein Quality Control and Apoptosis:
Proper folding of proteins is essential for the functional integrity of the cells. Consequently, the cells have devised sophisticated mechanisms to ensure proper “protein homeostasis” (Rutkowski, D. T. and Kaufman, R. J. 2004. Trends Cell Biol 14, 20-8). Accumulation of misfolded proteins at the endoplasmic reticulum (ER), also known as ER stress, activates a cellular adaptive response referred to as UPR (Unfolded Protein Response). ER stress is implicated in the pathophysiology of multiple neurodegenerative diseases, including Huntington's Disease and Alzheimer's (Oakes, S. A. et al., 2006. Curr Mol Med 6, 99-109). The BCL-2 family proteins BAX and BAK were recently shown to be required for the proper execution of the signaling pathways involved in UPR by physically associating with several components of this pathway (Hetz, C. et al., 2006. Science 312:572-6). It has been proposed that BAX/BAK serve to link UPR at the ER with the core apoptotic machinery at the mitochondria and thus orchestrate the cellular response to misfolded proteins through either proper execution of an adaptive response or cell death.
(iv) BCL-2 Family Proteins and Regulation of Mitochondrial Morphology During Life and Death:
Mitochondrial shape and reticular structure is dynamically regulated by fusion and fission processes (Griparic, L. and Van der Bliek, A. M. 2001. Traffic 2, 235-44). This ensures exchange of material between different mitochondria and elimination of those organelles unfit for function. The dynamic changes in mitochondrial morphology directly impact the metabolic fitness of the cell. During apoptosis, mitochondria undergo fragmentation prior o caspase activation (Frank, S. et al., 2001. Dev Cell 1, 515-25). Several BCL-2 family proteins are implicated in these processes. Anti-apoptotic molecules BCL-2/BCL-XL have pro-fusion activity that seems to be preserved throughout evolution. This coincides with their capacity to bind mitofusion-2 (Mfn-2), a protein known to govern mitochondrial fusion (Delivani, P. et al., 2005. Mol Cell 21, 761-73). The pro-apoptotic BAX regulate the activity of Mfn-2 directly (Karbowski, M. et al., 2006. Nature 443, 658-62). It is noteworthy that the role of BCL-2 family members in mitochondrial dynamics is also essential in healthy cells that have not received a death stimulus. It has been suggested that the BH3 domain of BAX is required for regulation of mitochondrial dynamics (Karbowski, M. et al., 2006. Nature 443, 658-62). Thus the same domain in BAX has the capacity to regulate two distinct functions; apoptosis and mitochondrial shape.
Glucose Homeostasis, Diabetes and the Metabolic Syndrome
Type 2 diabetes mellitus (T2DM) is a multigenetic disease that includes multiple metabolic abnormalities that commonly manifest in a failed glucose tolerance. The basic physiological tenets of T2DM include abnormalities in insulin production and function (Saltiel, A. R. and Kahn, C. R. 2001. Nature 414, 799-806). This involves changes in the function of pancreas, muscle, fat and liver. Pancreatic β-cells and hepatocytes are the main glucose sensors within the body (Accili, D. 2004. Diabetes 53, 1633-42). In response to blood glucose fluctuation, β-cells secrete insulin in a dose responsive manner. Insulin in turn stimulates glucose uptake by peripheral tissues such as muscle and fat and prompts proper storage of glucose as glycogen in the liver. Pancreatic islets are also prone to adapt their mass in order to meet the insulin secretory demands in the body (Bell G. I. and Polonsky K. S., 2001. Nature 414, 788-91; Weir G. C. et al., 2001. Diabetes 50 Suppl 1, S154-9; DeFronzo R. A., 1988. Diabetes 37, 667-87; Accili D. et al., 2001. Curr Mol Med 1, 9-23). The lack of proper glucose sensing and/or mass adaptation by islets contribute to T2DM. Hepatocytes sense fluctuations in blood glucose and adjust their function to either produce glucose during fasting, which helps keep adequate glucose supply to the brain, or to store glucose as glycogen when blood glucose levels exceed their normal range (Cherrington, A. D. 1999. Diabetes 48, 1198-1214). In addition to the regulation of carbohydrate metabolism, insulin also impacts fat metabolism by suppressing lipolysis in fat cells (DeFronzo, R. A. 2004. Int J Clin Pract Suppl 143, 9-21). Insulin resistance is a state in which muscle, fat and liver are insensitive to the action of insulin. Metabolic syndrome is defined as a cluster of metabolic deficiencies that include insulin resistance, dyslipidemia (including abnormal levels of plasma triglycerides), obesity and diabetes (Reaven, G. M. 2004. Diabetes Care 27, 1011-12).
Glucose Metabolism and Cancer
Individual cells depend on the availability of growth/survival factors that characteristically regulate both cellular metabolism and cell survival (Plas, D. R. and Thompson, C. B. 2002. Trends Endocrinol Metab 13, 75-8). Cellular metabolism is a term used to describe a group of chemical reactions that take place in a living cell or organism where nutrients like glucose are broken down to yield energy for vital processes. Insulin, insulin-like-growth factor (IGF-1) and multiple cytokines transduce signals via PI3K through the serine/threonine kinase AKT and related kinases to regulate glucose transport and metabolism (Cheatham, B. and Kahn, C. R. 1995. Endocr Rev 16, 117-42). Signaling downstream of AKT impacts glucose metabolism by regulating the levels of glucose transporters (Glut 1 and Glut 4) (FIG. 3). Consequently, mice lacking AKT2 exhibit low levels of Glut4 and develop marked insulin resistance (Cho, H. et al., 2001. Science 292, 1728-31). A second mechanism whereby AKT regulates glucose metabolism is by stimulating recruitment of hexokinase (HK) to mitochondria (Robey, R. B. and Hay, N. 2006. Oncogene 25, 4683-96). Hexokinase is the enzyme that catalyzes the first step in glucose metabolism (glycolysis) converting glucose to glucose-6 phosphate. The molecular mechanism underlying mitochondrial localization of HK is not fully known. As mitochondria-associated hexokinase has immediate access to mitochondrial ATP and escapes product inhibition by glucose 6 phosphate (G6P), this may explain how activated AKT augments glucose-6 phosphorylating activity (Bustamante, E. and Pdersern, P. L. 1977. Proc Natl Acad Sci USA 74, 3735-39; Arora, K. K. and Pedersen, P. L. 1988. J Biol Chem 263, 17422-28). Activated AKT further promotes survival by stimulating expression of several anti-apoptotic proteins, such as BCL-XL and MCL-1 as well as phosphorylating several key cellular players, including Forkhead transcription factors, BAD and NFκB (Datta, S. R. et al., 1999. Genes Dev 13, 2905).
Resistance to apoptosis and increased cellular metabolism are common characteristics of tumors. BCL-2 was originally discovered due to its chromosomal translocation in follicular lymphoma. In addition, mouse models of BCL-2 family proteins clearly indicate that defects in apoptosis can be a primary oncogenic event (McDonnel, T. J. et al., 1989. Cell 57, 79-88; McDonnel, T. J. et al., Nature 349, 254-56). The chromosomal translocation in the Bcl-2 gene found in follicular lymphoma in humans juxtaposes the Bcl-2 coding sequence next to immunoglobulin (Ig) gene sequences. Importantly, recapitulating this chromosomal translocation using Bcl-2-Ig transgenic mice was sufficient to cause diffuse large-cell lymphoma over time. Likewise, several findings suggest that the pro-apoptotic BCL-2 family members may function as tumor suppressors in that their loss of function contributes to malignancy. Increased incidence of choroid plexus tumors in Bax-null mice expressing a truncated SV40 T antigen and of chronic myelomonocytic leukemia (CMML) in Bid-deficient mice reflect their importance in neuronal cell survival and myeloid homeostasis, respectively (Zinkel, S. et al., 2003. Genes Dev 17, 229-39; Yin, C. et al., 1997. Nature 385, 637-40). Bad-null mice progress to diffuse large B cell lymphoma (DLBCL) of germinal center origins (Ranger, A. M. et al., 2003. Proc Natl Acad Sci USA 100, 9324-29). This may reflect a potential role for BAD in regulating the cellular homeostasis of mature B cells as they migrate to germinal centers.
The relevance of cellular metabolism to malignancy was originally recognized by Warburg, who noted that tumors often display high glycolytic rates. Glycolysis accounts for ˜60% of ATP within tumor cells and provides metabolic intermediates for synthesis of macromolecules including nucleic acids needed for DNA synthesis and their rapid proliferation. Indeed several well characterized oncogenes in human cancers, including Ras, Myc, Akt are known to target the glycolytic pathway (Semenza, G. L. 2001. Novartis Found Symp 240, 251-60). Warburg's hypothesis further suggested that high glycolytic rates might be due to impaired mitochondrial respiration; however, this finding has proven somewhat variable in tumors. Recent studies have shown that even in the presence of fully functional oxidative phosphorylation (OXPHOS) capacity by mitochondria, tumor cells preferentially support their bioenergetic demands through glycolysis (Fantin, V. R. et al., 2006. Cancer Cell 9, 425-34). This switch is required for tumor maintenance as interference with glycolysis is associated with a compensatory increase in OXPHOS concomitant with a decline in proliferative capacity of tumor cells. Possible rationale for this “glycolytic switch” in tumors may be that in addition to providing ATP at a faster rate, glycolytic products (mainly pyruvate) are used as intermediates for synthesis of fatty acids (FIG. 3). This ensures that tumor cells have sufficient supply of fatty acids for new membrane synthesis to keep up with the high rate of cellular proliferation. Furthermore, reliance on glycolysis rather than OXPHOS ensures that tumors can grow in the absence of oxygen (hypoxia) prior to vascularization (Gatenby. R. A. and Gillies, R. J. 2004. Nat Rev Cancer 4, 891-99). In several human tumors, multiple key enzymes involved in glucose metabolism exhibit increased activity when compared to normal tissues. These include hexokinase (HK), phosphofructokinase (PFK), pyruvate kinase (PK) and lactate dehydrogenase (LDH). Mechanisms underlying increased activity have been best studied in the case of HK enzymes and include increased expression (secondary to gene amplification and/or promoter activation), increased binding to mitochondria and/or a switch in gene expression from high (hexokinase IV) to low km (hexokinase I-III) isoforms (Rempel, A, et al., 1994. Biochem J 303, 269-74; Klimek, F. et al., 1993. Carcinogenesis 14, 1857-61; Mazurek, S. et al., 1999. J cell Phyisol 181, 136-46). These observations underscore the importance of targeting glycolysis or the use of glycolytic intermediate in specific pathways in tumors as a promising therapeutic strategy.
Recent evidence also suggests that obesity and metabolic syndrome are associated with high risk of cancer, including colorectal cancer (Gunter, M. J. and Leizmann, M. F. 2006. J Nutr Biochem 17, 145-56), breast cancer (Lorincz, A. M. 2006. Endocr Relat Cancer 13, 279-92) and prostate cancer (O'Malley, R. L. and Taneja, S. S. 2006. Can J Urol Suppl 2, 11-7). Although, the molecular links between these metabolic abnormalities and cancer is not fully understood, several studies suggest that elevated levels of plasma insulin, as seen in insulin resistant state, activates cellular proliferation in epithelial cells. Furthermore, insulin can increase the levels of Insulin-like Growth Factor 1 (IGF-1), a growth hormone with significant proliferative and anti apoptotic activity (Cowey, S, and Hardy, R. W. 2006. Amer J Pathol 169, 1505-22). Insulin and IGF-1 also regulate the sex steroids, which in turn modulate the activity of estrogen and androgens and consequently development of cancers dependent on sex hormones, including breast and prostate cancers (Calle, E. E. and Kaaks, R. 2004. Nat Rev Cancer 4, 579-91). Furthermore, hormones produced by fat cells, adipokines, have proliferative, angiogenic and pro-inflammatory effects. Adipokines influence cancer cells either directly through these effects or indirectly by causing insulin resistance (and thus hyperinsulinemia) (Cowey, S, and Hardy, R. W. 2006. Amer J Pathol 169, 1505-22).